14-3-3 alpha+beta Recombinant Rabbit Monoclonal Antibody [SD0837]
cat.: ET1612-99
Product Type: Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity: Human, Mouse, Rat
Applications: WB, IF-Cell, IHC-P, FC, IP
Clonality: Monoclonal
Clone number: SD0837
Form: Liquid
Storage condition: Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.
Storage buffer: 1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration: 1ug/ul
Purification: Protein A affinity purified.
Molecular weight: Predicted band size: 28 kDa
Isotype: IgG
Immunogen: Synthetic peptide corresponding to C terminal Human 14-3-3 alpha + beta aa 197-246 / 246.
Positive control: Neuro-2a cell lysate, C6 cell lysate, mouse brain tissue lysate, rat brain tissue lysate, Hela cell lysate, HepG2 cell lysate, 293T cell lysate, SK-Br-3 cell lysate, Hela, A431, Neuro-2a , human breast carcinoma tissue, mouse brain tissue, mouse skin tissue, human breast tissue, rat brain tissue.
Subcellular location: Cytoplasm, Melanosome.
Recommended Dilutions:
  WB
  IF-Cell
  IHC-P
  FC
  IP
1:500-1:2,000
1:100-1:500
1:50-1:200
1:1,000
Use at an assay dependent concentration.
Uniprot #: SwissProt: P31946 Human | Q9CQV8 Mouse | P35213 Rat
Alternative names: 14 3 3 alpha 14 3 3 protein beta/alpha 14-3-3 protein beta/alpha 1433B_HUMAN Brain protein 14 3 3 beta isoform GW128 HS 1 KCIP-1 KCIP1 N-terminally processed Protein 1054 Protein kinase C inhibitor protein 1 YWHAA YWHAB
Images
ET1612-99_1.jpg Fig1: Western blot analysis of 14-3-3 alpha+beta on different lysates with Rabbit anti-14-3-3 alpha+beta antibody (ET1612-99) at 1/2,000 dilution.

Lane 1: Neuro-2a cell lysate (20 µg/Lane)
Lane 2: C6 cell lysate (20 µg/Lane)
Lane 3: Mouse brain tissue lysate (40 µg/Lane)
Lane 4: Rat brain tissue lysate (40 µg/Lane)

Predicted band size: 28 kDa
Observed band size: 28 kDa

Exposure time: 14 seconds; ECL: K1801;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1612-99) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
ET1612-99_2.jpg Fig2: Western blot analysis of 14-3-3 alpha+beta on different lysates with Rabbit anti-14-3-3 alpha+beta antibody (ET1612-99) at 1/1,000 dilution.

Lane 1: Hela cell lysate
Lane 2: HepG2 cell lysate
Lane 3: 293T cell lysate
Lane 4: SK-Br-3 cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 28 kDa
Observed band size: 28 kDa

Exposure time: 1 minute;

12% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1612-99) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:300,000 dilution was used for 1 hour at room temperature.
ET1612-99_3.jpg Fig3: ICC staining of 14-3-3 alpha+beta in Hela cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1612-99, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
ET1612-99_4.jpg Fig4: ICC staining of 14-3-3 alpha+beta in A431 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1612-99, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
ET1612-99_5.jpg Fig5: Immunocytochemistry analysis of Neuro-2a cells labeling 14-3-3 alpha+beta with Rabbit anti-14-3-3 alpha+beta antibody (ET1612-99) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-14-3-3 alpha+beta antibody (ET1612-99) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
ET1612-99_6.jpg Fig6: Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using anti-14-3-3 alpha+beta antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1612-99, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1612-99_7.jpg Fig7: Immunohistochemical analysis of paraffin-embedded mouse brain tissue using anti-14-3-3 alpha+beta antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1612-99, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1612-99_8.jpg Fig8: Immunohistochemical analysis of paraffin-embedded mouse skin tissue using anti-14-3-3 alpha+beta antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1612-99, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1612-99_9.jpg Fig9: Immunohistochemical analysis of paraffin-embedded human breast tissue using anti-14-3-3 alpha+beta antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1612-99, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1612-99_10.jpg Fig10: Immunohistochemical analysis of paraffin-embedded rat brain tissue with Rabbit anti-14-3-3 alpha+beta antibody (ET1612-99) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1612-99) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1612-99_11.jpg Fig11: Flow cytometric analysis of HeLa cells labeling 14-3-3 alpha+beta.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1612-99, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
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